Wistar kyoto rats

The procedures involving animals were conducted according to national and international regulations (Directive 2010/63/EU), and appropriate measures were used to minimize their pain or discomfort. The Ethics Committee of the University of Padova (OPBA) and the Italian Ministry of Health reviewed and approved all the protocols (Prot. no. 721, 2017), which fulfill completely the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines [20 (link)].
In this study, 16 male Wistar Kyoto rats (Charles River, Boston, MA, USA) were randomly assigned to two experimental groups: One group (standard diet rats, controls) consisted of animals (n = 8) fed with the standard diet used in the animal facility, whereas the others (Western diet rats, n = 8) were fed with a diet rich in fat (60/Fat, kcal from: 23.5% protein, 18.4% carbohydrate; 60.3% fat; Harlan Laboratories, Lesmo (Monza Brianza) Italy), boosted with 30% fructose in drinking water, as reported elsewhere [21 (link),22 (link)]. Their body weight was measured once a week. After 12 weeks, the rats were sacrificed, and blood samples and livers were collected.

A total of 32 adult (approximately 6 month-old) male GK rats, bred at Wilfrid Laurier University from stock originally obtained from Charles River Canada (St. Constance, QC) and 30 age-matched Wistar Kyoto rats (Charles River Canada, St. Constance, QC) were used in this experiment. All animals were housed individually with ad libitum access to food and water on a 12:12-h reverse light cycle. All procedures were approved by the Wilfrid Laurier University Animal Care Council according to the guidelines outlined by the Canadian Council on Animal Care. Subjects engaged in one of 2 tasks: (a) CMT (Dere et al., 2005a ,b (link); Kart-teke et al., 2006 (link)), or (b) environmental exploration (Guzowski et al., 1999 (link)). Each of these is described below. An additional caged control group (n = 6 rats per strain) was sacrificed directly from the home cage as a negative control.

Twelve‐week‐old female Wistar–Kyoto rats (Charles River Labs) were used for in vivo experimental protocols. To induce I/R injury, rats were provided general anesthesia and then a thoracotomy was performed at the fourth intercostal space to expose the heart and left anterior descending coronary artery (LAD). A 7‐0 silk suture was then used to ligate the LAD, which was subsequently removed after 45 min to allow for reperfusion. Ten minutes later, 100 μl of EV‐YF1, Ys or vehicle was injected into the LV cavity over a period of 20 seconds with aortic cross‐clamp. Briefly, 10 μg of EV‐YF1 or Ys was incubated in IMDM basal media (Thermo Scientific) with Dharmafect transfection reagent (Dharmacon) for 10 min at room temperature then resuspended in 100 μl IMDM for injection.

Male spontaneously hypertensive rats (SHR/NCrl) and normotensive Wistar-Kyoto rats (WKYs) in four age groups were obtain from Charles River Laboratories and housed in controlled 12 h light/dark conditions, in a constant temperature of 21 °C, with ad libitum access to water and food. Part of the animals were randomly assigned to groups treated for 30 days with GKT137831 (CAS 1218942-37-0, Adooq Bioscience LLC, USA), ML171 (CAS 6631-94-3, Merck, Germany) or placebo mixed in food (both at 60 mg/kg/day [18 (link)]). Blood pressure was measured using non-invasive tail cuff plethysmography using BP2000 Blood Pressure Analysis System (Visitech Systems Inc.), following 7 days of training period as described before [26 (link)]. At the end of experiment, rats were heparinised and euthanized using CO₂ inhalation and perfused via the left ventricle of the heart using cold PBS to ensure blood cells are not contributing to organ flow cytometric analysis. All in vivo work was performed in accordance with the Animals Scientific Procedures Act 1986 and ARRIVE (Animal Research: Reporting of In Vivo Experiments) Guidelines and approved by Jagiellonian University Ethics Committee (107/2014). All experiments conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.